This application proposes studies to identify the mechanism by which the Helicobacter pylori vacuolating cytotoxin (VacA) is trafficked to mitochondria in host epithelial cells, where the toxin induces mitochondrial damage. VacA binds to the surface of host cells and is internalized into the endolysosomal system, but a major gap in knowledge to be addressed in this proposal is the mechanism by which the toxin targets and localizes to mitochondria. We and others have failed to detect VacA within the host cell cytosol, suggesting that this membrane-interacting, pore-forming toxin is not taken up into mitochondria using existing pathways used by endogenous proteins that are imported from the cytosol. Rather, our current model predicts that VacA is transported from the cell surface to mitochondria by vesicular trafficking. Given that vesicular trafficking pathways from the cell-surface to mitochondria have not previously been identified, this application proposes studies to evaluate the hypothesis that VacA-containing vesicles (VCVs) are dynamically re-modeled from early- endosomal-like compartments to trafficking vesicles that are competent for targeting mitochondria. Working in the laboratory of Dr. Steven Blanke at the University of Illinois at Urbana-Champaign, I will test the prediction of this hypothesis using a set of studies proposed within 2 Specific Aims: 1) In Aim 1, studies are proposed to test the prediction that, if VCVs are dynamically remodeled, the proteome of VCVs will change as a function of time to become enriched with proteins that mediate mitochondrial targeting and/or vesicular trafficking. In support of the proposed studies, I have developed and optimized new approaches for magnetically isolating VCVs enriched in VacA labeled with ferromagnetic nano-particles. 2) In Aim 2, I will test the prediction that if VCVs become progressively enriched with cellular proteins required for mitochondrial targeting, then knockdown or knockout of these cellular proteins by RNA interference or gene deletion will result in decreased or blocked VacA localization to mitochondria, and, VacA-mediated disruption of mitochondrial function. Completion of the proposed studies will address a major gap in our understanding of cellular intoxication by VacA, which is a major virulence factor of H. pylori, a pathogen of significant health concern given human infection increases the risk for the development of gastric cancer. These studies will also reveal, potentially for the first time, an intracellular trafficking mechanism by which proteins move from the surface of host cells to the mitochondria, which may be relevant to the broader class of mitochondrial-acting, pore-forming toxins generated by clinically relevant pathogenic bacteria. Identifying the mechanisms and importance of bacterial toxin-mediated modulation of host cells is an important step towards developing therapies for blocking toxin activities that contribute to pathogenesis.